Binding specificity

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there is no a clear association to the protein, the figure 6 shows a typical aspecific sensorgram with a low millimolar dissociation value. This result probably suggest the importance of whole sequence, 38-mer peptide, for fibronectin binding.

Figure 6 Binding of 13-mer peptide and fibronectin protein

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adsorption to fibronectin coated substrate with higher concentration, therefore lower peptide concentration released.

Figure 7 Reverse-phase-HPLC of 6-mer peptide specific binding on fibronectin coated glass. The chromatograms performed at 280 nm shows un bound peptide fractions at three different fibronectin layers. Black peak shows the 6-mer peptide release after incubation on 10 μg/mL fibronectin layer.

unlike blue and green peaks show unbound peptide after binding with 30 and 50 μg/mL protein layers, respectively.

4.3.4 PEI-peptide conjugate characterization

The conjugation of the hexapeptide (GGWSHW) to linear PEI 25 kDa was confirmed by ¹H-NMR spectroscopy. Wang et al. reported that PEI metylen peak shifts from 2.5 to 3 ppm when it forms an amidic bond with the activated amino terminal group of the peptide. NMR spectrum of peptide-PEI is shown in fig. 8. A very intense peak is found at 3 ppm confirming the covalent binding of PEI to the peptide. Signals at around 10 ppm are typical of Trp indolic protons where signals in the region from 8 to6 ppm become to peptide backbond amide and side chains aromatic protons.

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Figure 8 NMR spectra of PEI-6-mer and PEI-peptide complex. The shift of PEI peak (B) from 2.7 to 3.2 ppm confirms the correct covalent bond between peptides and polymer, the signals at 10 ppm and at 8-6 ppm typical of Trp residues and peptide backbone amide and side chains are visible in the spectrum (A).

A

B

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The correct PEI-Peptide conjugation was confirmed by RP-HPLC, as well. In the figure 9 PEI-peptide crude complex chromatogram was reported. The presence of a non sharp peak at 230 nm that covers tryptophan peak at 280nm indicates the correct complex formation for both peptides.

Figure 9 RP-HPLC analysis of PEI-peptide complex. a) 6-mer complex at 280 nm in blue and at 220 nm in black. The presence of a non sharpe peak at 220 nm confirms the correct formation of the complex that is not visible at 280 nm.

4.3.5 Imaging of PEI/DNA complexes specifically adsorbed to fibronectin coated substrates and adhesive/transfective islands

Results of immunofluorescence on samples with cells seeded on PEI/DNA complexes specifically adsorbed to fibronectin coated substrates and fibronectin stamps to glass slides such as adhesive islands were shown in the figure 10 and 11.

Figures show blue spots of PEI/DNA complexes adsorbed to green layer/spots of fibronectin and cell seeded on this system with red cytoskeleton.

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Figure 10 CLSM image of cell on complexes specifically adsorbed to fibronectin coated substrate through linker 6-mer peptide

Figure 11 CLSM image of array of printed fibronectin spots. 3×3 array of 500 um diameter islands of fluorescently labeled IgG generated by contact printing

250 μm

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